Selective Reconstitution of Gastrin-Releasing Peptide Receptor with Gα q

• Published in: Proceedings of the National Academy of Sciences - PNAS 1997-01, Vol.94 (2), p.751-756
• Main Authors: Hellmich, Mark R., Battey, James F., Northup, John K.
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Agonists; Animals; Biological Sciences; Cell lines; Cell Membrane - metabolism; Cell membranes; Cell-Free System; Cells; Drug interactions; Enzyme Activation; Gastrin-Releasing Peptide; GTP-Binding Proteins - antagonists & inhibitors; GTP-Binding Proteins - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Guanosine Triphosphate - metabolism; Ligands; Mice; P branes; Peptides - metabolism; Pertussis Toxin; Receptors; Receptors, Bombesin - metabolism; Recombinant Proteins; Signal Transduction; Transfection; Ungulates; Virulence Factors, Bordetella - pharmacology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.2.751

Identification of the molecular mechanisms that determine specificity of coupling interactions between gastrin-releasing peptide receptors (GRPrs) and their cognate heterotrimeric GTP-binding proteins is a fundamental step in understanding the signal transduction cascade initiated by receptor-ligand interaction. To explore these mechanisms in greater detail, we have developed an in situ reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTPγ S to purified G protein α subunits. Binding studies with 125I-labeled [D-Tyr6] bombesin (6-13) methyl ester (125I-Tyr-ME), a GRPr specific antagonist, show a single binding site with a Kd = 1.4 nM ± 0.4 (mean ± SD, n = 3) and capacity of 15-22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2- to 3-fold enrichment of binding sites compared with the membranes before extraction. Quantitative ligand displacement analysis using various unlabeled GRPr agonists shows a rank order of potency characteristic of the GRPr: bombesin ≥ GRP ≫ neuromedin B. Reconstitution of urea extracted membranes with a purified Gα q showed that receptor-catalyzed binding of GTPγ S was dependent on agonist (GRP) and Gβ γ subunits. The EC50 for GRP was 3.5 nM, which correlates well with the reported Kd of 3.1 nM for GRP binding to GRPr expressed in mouse fibroblasts [Benya, R. V., et al. (1994) Mol. Pharmacol. 46, 235-245]. The apparent Kd for bovine brain Gβ γ in this assay was 60 nM, and the Km for squid retinal Gα q was 90 nM. The GRPr-catalyzed binding of GTPγ S is selective for Gα q, since we did not detect receptor-catalyzed exchange using either Gα i/o or Gα t. These data demonstrate that GRPr can functionally couple to Gα q but not to the pertussis toxin-sensitive Gα i/o or retinal specific Gα t. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.